Field of the Invention
This invention relates to systems and methods for imaging sample materials within a microfluidic device during an assay reaction process.
Discussion of the Background
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying DNA.
With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of the DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
In some applications, it is important to monitor the accumulation of DNA products as the amplification process progresses. Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the amplification process over time allows one to determine the efficiency of the process, as well as estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Further examples of systems, methods, and apparatus for high throughput approaches to performing PCR and other amplification reactions are described in the following publications that are related to the subject matter of the present disclosure.
U.S. Patent Application Publication No. 2008/0176230 to Owen et al. entitled “Systems and methods for real-time PCR” (the '230 publication”), the disclosure of which is hereby incorporated by reference, describes systems and methods for the real-time amplification and analysis of a sample of DNA within a micro-channel.
U.S. Pat. No. 7,629,124 to Hasson et al. entitled “Real-time PCR in micro-channels” (the '124 patent”) the disclosure of which is hereby incorporated by reference, describes systems and methods for performing real time PCR in micro-channels by continuously moving boluses of test solution separated by carrier fluid through the micro-channels and performing a process, such as PCR, on each bolus and measuring signals, such as fluorescent signals, at different locations along a defined section of the channel.
U.S. Pat. No. 7,593,560 to Hasson et al. entitled “Systems and methods for monitoring the amplification and dissociation behavior of DNA molecules” (the '560 patent”), the disclosure of which is hereby incorporated by reference, describes the use of sensors for monitoring reactions within microfluidic channels. The sensor has a defined pixel array for collecting image data, and image data from a select window of pixels (a sub-set of the entire array), which encompasses a portion of interest of a micro-channel, is processed and stored for each of the micro-channels.
Once there are a sufficient number of copies of the original DNA molecule, the DNA can be characterized. One method of characterizing the DNA is to examine the DNA's dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA) with increasing temperature. The process of causing DNA to transition from dsDNA to ssDNA is sometimes referred to as a “high-resolution temperature (thermal) melt (HRTm)” process, or simply a “high-resolution melt” process.
To monitor a PCR process and/or a melting process (quantitatively and/or qualitatively), an imaging system may be employed to measure an optically detectable characteristic, such as fluorescence, of a dye that is incorporated into the sample material and that varies in a detectable manner as the number of copies of the original DNA molecule increases and/or as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA) with increasing temperature. The accuracy and reliability of nucleic acid assays depends, to a large extent, on the accuracy and precision of such imaging systems. Moreover, the costs of such imaging systems are a significant portion of the cost of an overall instrument for performing nucleic acid assays.
Thus, there is a continuing need for improvements in accuracy, precision, and cost effectiveness of imaging systems for monitoring nucleic acid diagnostic assays and other biological processes.